Gene expression in diseased and healthy individuals is oftentimes different and characterizable. The ability to monitor gene expression in such cases provides medical professionals with a powerful diagnostic tool. This form of diagnosis is especially important in the area of oncology, where it is thought that the overexpression of an oncogene, or the underexpression of a tumor suppressor gene, results in tumorogenesis. See Mikkelson et al. J. Cell. Biochem. 1991, 46, 3-8.
One can indirectly monitor gene expression, for example, by measuring a nucleic acid (e.g., mRNA) that is the transcription product of a targeted gene. The nucleic acid is chemically or biochemically labeled with a detectable moiety and allowed to hybridize with a localized nucleic acid probe of known sequence. The detection of a labeled nucleic acid at the probe position indicates that the targeted gene has been expressed. See International Application Publication Nos. WO 97/27317, WO 92/10588 and WO 97/10365.
The labeling of a nucleic acid is typically performed by covalently attaching a detectable group (label) to either an internal or terminal position. Scientists have reported a number of detectable nucleotide analogues that have been enzymatically incorporated into an oligo- or polynucleotide. Langer et al., for example, disclosed analogues of dUTP and UTP that contain a covalently bound biotin moiety. Proc. Natl. Acad. Sci. USA 1981, 78, 6633-6637. The analogues, shown below, possess an allylamine linker arm that is attached to the C-5 position of the pyrimidine ring. The dUTP and UTP analogues, wherein R is H or OH, were incorporated into a polynucleotide. 
Petrie et al. disclosed a dATP analogue, 3-[5-[(N-biotinyl-6-aminocaproyl)-amino]pentyl]-1-(2-deoxy-xcex2-D-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine-5xe2x80x2-triphosphate. Bioconjugate Chem. 1991, 2, 441-446. The analogue, shown below, is modified at the 3-position with a linker arm that is attached to a biotin moiety. Petrie et al. reported that the compound wherein R is biotin is incorporated into DNA by nick translation. 
Prober et al. disclosed a set of four dideoxynucleotides, each containing a succinylfluorescein dye. Science 1987, 238, 336-341. The dideoxynucleotides, one of which is shown below, were enzymatically incorporated into an oligonucleotide through a template directed extension of a primer. The compounds provided for a DNA sequencing method based on gel migration. 
Herrlein et al. disclosed modified nucleoside trisphosphates of the four DNA bases. Helv. Chim. Acta 1994, 77, 586-596. The compounds, one of which is shown below, contain a 3xe2x80x2-amino group containing radioactive or fluorescent moieties. Herrlein et al. further described the use of the nucleoside analogues as DNA chain terminators. 
Cech et al. disclosed 3xe2x80x2-amino-functionalized nucleoside triphosphates. Collect. Czech. Chem. Commun. 1996, 61, S297-S300. The compounds, one of which is shown below, contain a fluorescein attached to the 3xe2x80x2-position through an amino linker. Cech et al. proposed that the described functionalized nucleosides would be useful as terminators for DNA sequencing. 
The present invention relates to nucleic acid labeling compounds. More specifically, the invention provides heterocyclic derivatives containing a detectable moiety. The invention also provides methods of making such heterocyclic derivatives. It further provides methods of attaching the heterocyclic derivatives to a nucleic acid.
The development of a novel nucleic acid labeling compound that is effectively incorporated into a nucleic acid to provide a readily detectable composition would benefit genetic analysis technologies. It would aid, for example, in the monitoring of gene expression and the detection and screening of mutations and polymorphisms. Such a compound should be suitable for enzymatic incorporation into a nucleic acid. Furthermore, the nucleic acid to which the labeling compound is attached should maintain its ability to bind to a probe, such as a complementary nucleic acid.
The present invention provides nucleic acid labeling compounds that are capable of being enzymatically incorporated into a nucleic acid. The nucleic acids to which the compounds are attached maintain their ability to bind to a complementary nucleic acid sequence.
The nucleic acid labeling compounds of the present invention are of the following structure:
A-Oxe2x80x94CH2-T-Hc-L-(M)m-Q 
wherein A is hydrogen or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; T is a template moiety; Hc is a heterocyclic group; L is a linker moiety; Q is a detectable moiety; and M is a connecting group, wherein m is an integer ranging from 0 to about 5.
In one embodiment, the nucleic acid labeling compounds have the following structures: 
wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid;
X is O, S, NR1 or CHR2, wherein R1 and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is is amido alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
In another embodiment, A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94C(O)NH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 2 to about 10; Q is biotin or a carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94C(O)NH(CH2)4NHxe2x80x94; Q is biotin; and, M is xe2x80x94CO(CH2)5NH, wherein m is 1.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94C(O)NH(CH2)4NHxe2x80x94; Q is 5-carboxyfluorescein; and, m is 0.
In one embodiment, the nucleic acid labeling compounds have the following structures: 
wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NR1 or CHR2, wherein R1 and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is amino alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
In another embodiment, A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94NH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 2 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94NH(CH2)4NHxe2x80x94; Q is biotin; and, m is 0.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94NH(CH2)4NHxe2x80x94; Q is 5-carboxyfluorescein; and, m is 0.
In one embodiment, the nucleic acid labeling compounds have the following structures: 
wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NR1 or CHR2, wherein R1 and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is alkynyl alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
In another embodiment, A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94Cxe2x89xa1C(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94Cxe2x89xa1CCH2NHxe2x80x94; Q is biotin; and, m is 1.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94Cxe2x89xa1CCH2NHxe2x80x94; Q is 5-carboxyfluorescein; and, m is 1.
In one embodiment, the nucleic acid labeling compounds have the following structures: 
wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NR1 or CHR2, wherein R1 and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is amino alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
In another embodiment, A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94NH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 2 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94NH(CH2)4NHxe2x80x94; Q is biotin; and, M is xe2x80x94CO(CH2)5NHxe2x80x94, wherein m is 1.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94NH(CH2)4NHxe2x80x94; Q is 5-carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94, wherein m is 1.
In one embodiment, the nucleic acid labeling compounds have the following structures: 
wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NR1 or CHR2, wherein R1 and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is functionalized alkyl, alkenyl alkyl or alkynyl alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
In another embodiment, A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94CHxe2x95x90CH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94CHxe2x95x90CHCH2NHxe2x80x94; Q is biotin; and, m is 0.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94CHxe2x95x90CHCH2NHxe2x80x94; Q is 5-carboxyfluorescein; and, m is 0.
In one embodiment, the nucleic acid labeling compounds have the following structures: 
wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NR1 or CHR2, wherein R1 and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is functionalized alkyl, alkenyl alkyl or alkynyl alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
In another embodiment, A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94CHxe2x95x90CH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94CHxe2x95x90CHCH2NHxe2x80x94; Q is biotin; and, m is 0.
In another embodiment, Y is H or OH; Z is H or OH; L is xe2x80x94CHxe2x95x90CHCH2NHxe2x80x94; Q is 5-carboxyfluorescein; and, m is 0.
The present invention also provides nucleic acid derivatives produced by coupling a nucleic acid labeling compound with a nucleic acid and hybridization products comprising the nucleic acid derivatives bound to a complementary probe.
In one embodiment, the nucleic acid labeling compounds used in the coupling have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkly or aryl; L is xe2x80x94C(O)NH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 2 to about 10; Q is biotin or a carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94, wherein mn is 1 or 0.
The hybridization product formed from this nucleic acid derivative comprises the nucleic acid derivative bound to a complementary probe. In one embodiment, the probe is attached to a glass chip.
In another embodiment, the nucleic acid labeling compounds used in the coupling have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94NH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 2 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The hybridization product formed from this nucleic acid derivative comprises the nucleic acid derivative bound to a complementary probe. In one embodiment, the probe is attached to a glass chip.
In another embodiment, the nucleic acid labeling compounds used in the coupling have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94Cxe2x89xa1C(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The hybridization product formed from this nucleic acid derivative comprises the nucleic acid derivative bound to a complementary probe. In one embodiment, the probe is attached to a glass chip.
In another embodiment, the nucleic acid labeling compounds used in the coupling have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94NH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 2 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The hybridization product formed from this nucleic acid derivative comprises the nucleic acid derivative bound to a complementary probe. In one embodiment, the probe is attached to a glass chip.
In another embodiment, the nucleic acid labeling compounds used in the coupling have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94CHxe2x95x90CH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The hybridization product formed from this nucleic acid derivative comprises the nucleic acid derivative bound to a complementary probe. In one embodiment, the probe is attached to a glass chip.
In another embodiment, the nucleic acid labeling compounds used in the coupling have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94CHxe2x95x90CH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The hybridization product formed from this nucleic acid derivative comprises the nucleic acid derivative bound to a complementary probe. In one embodiment, the probe is attached to a glass chip.
The present invention also provides methods of synthesizing nucleic acid derivatives by attaching a nucleic acid labeling compound to a nucleic acid. It further provides methods of detecting nucleic acids involving incubating the nucleic acid derivatives with a probe.
In one embodiment, the nucleic acid labeling compounds attached to the nucleic acid have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94C(O)NH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 2 to about 10; Q is biotin or a carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The method of nucleic acid detection using the nucleic acid derivative involves the incubation of the derivative with a probe. In one embodiment, the probe is attached to a glass chip.
In one embodiment, the nucleic acid labeling compounds attached to the nucleic acid have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94NH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 2 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The method of nucleic acid detection using the nucleic acid derivative involves the incubation of the derivative with a probe. In one embodiment, the probe is attached to a glass chip.
In one embodiment, the nucleic acid labeling compounds attached to the nucleic acid have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94Cxe2x89xa1C(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The method of nucleic acid detection using the nucleic acid derivative involves the incubation of the derivative with a probe. In one embodiment, the probe is attached to a glass chip.
In one embodiment, the nucleic acid labeling compounds attached to the nucleic acid have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94NH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 2 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The method of nucleic acid detection using the nucleic acid derivative involves the incubation of the derivative with a probe. In one embodiment, the probe is attached to a glass chip.
In one embodiment, the nucleic acid labeling compounds attached to the nucleic acid have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94CHxe2x95x90CH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The method of nucleic acid detection using the nucleic acid derivative involves the incubation of the derivative with a probe. In one embodiment, the probe is attached to a glass chip.
In one embodiment, the nucleic acid labeling compounds attached to the nucleic acid have the following structures: 
wherein A is H or H4O9P3xe2x80x94; X is O; Y is H or OR9, wherein R9 is H, alkyl or aryl; Z is H, N3, F or OR10, wherein R10 is H, alkyl or aryl; L is xe2x80x94CHxe2x95x90CH(CH2)nNHxe2x80x94, wherein n is an integer ranging from about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is xe2x80x94CO(CH2)5NHxe2x80x94 or xe2x80x94CO(CH2)5NHCO(CH2)5NHxe2x80x94, wherein m is 1 or 0.
The method of nucleic acid detection using the nucleic acid derivative involves the incubation of the derivative with a probe. In one embodiment, the probe is attached to a glass chip.